Compositions and methods for detecting and treating hiv infections

ABSTRACT

HIV infection can be detected by measuring phosphorylation levels of the actin-depolymerizing factor (AFD)/cofilin family, and infection can be treated and/or prevented by modulating the HIV co-receptor signaling pathway.

INFORMATION ON RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/744,776, filed on May 4, 2007, which claims the priority benefit ofU.S. Provisional Application No. 60/797,745 filed on May 5, 2006, andU.S. Provisional Application No. 60/894,150 filed on Mar. 9, 2007, allof which are hereby incorporated herein by reference.

STATEMENT REGARDING SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberA1069981 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

HIV infection of susceptible cells, such as CD4+ T-cells, is mediated bythe interaction of CD4 and a cell surface chemokine co-receptor with thegp120 envelope protein. The HIV viral particle initially binds via itsgp120 envelope protein to the CD4 receptor of the target cell. Aconformational change occurs in gp120 that results in its subsequentbinding to a chemokine receptor, such as CCR5. See, e.g., Wyatt et al.,Science, 280:1884-1888 (1998). HIV-1 isolates arising subsequently inthe infection bind to the CXCR4 chemokine receptor.

HIV does not immediately replicate in all infected cells, but insteadcan progress to a state of latent infection, where the HIV is dormant.Upon activation of a latent infected cell, for example by engagingT-cell surface CD3 and CD28, the “dormant” HIV virus can becomeactivated, initiating the process of viral replication. HIV replicationresults in the production of infectious HIV particles, facilitating thespread of the infection throughout the subject cells.

The pool of latently infected cells in the resting CD4+ T-cellcompartment is considered one of the major impediments to HIVeradication. When latently infected, resting T-cells become reactivated,viral particles released during the reactivation process can spread toand infect resting T-cells, as well as activated CD4+ T-cells. Thisreactivation process can facilitate the continual replenishment of theCD4+ T-cell reservoir, offsetting the benefits of antiviral therapy,such as HAART, and contributing to the persistence of HIV and initiationof new infection cycles. See, e.g., Chun et al., J. Clin. Invest.115:3250-3255, 2005.

Accordingly, methods capable of detecting and treating HIV infectionduring its latent phase are needed.

SUMMARY

In one aspect, there is provided a method for detecting HIV infection ina patient, comprising measuring the phosphorylation level of cofilin ina sample from the patient. In one embodiment, the phosphorylation levelis measured for serine-3. In another embodiment, the measuring isconducted by detecting a phosphorylation-driven conformational change onelectron transport.

In another aspect, there is provided a method for treating and/orpreventing HIV infection in a patient, comprising: administering to saidpatient an agent that inhibits HIV trigger receptor signaling; inhibitsactin depolymerization; enhances the assembly of actin; stabilizes actinfilaments; induces polymerization of monomeric actin; binds to F-actinor cofilin; or inhibits actin and cofilin activity.

In one embodiment, the agent is selected from the group consisting ofjasplakinolide, PP2A, PP1, slingshot phosphatases, FR225659, fostriecin,calyculin A, cantharidin, jasplakinolide; phaloidin; chondramides,chondramide A, B, C, and D; (−)-doliculide; dolastatin-11; dolastatin3-Nor; Majusculamide; dolastatin Hmp;alpha-cyano-3,4-dihydroxy-N-benzylcinnamide (AG490); 1,2,3,4,5,6-;JSI-124; benzylidenemalonitriles (“tyrphostins”); WHI-P154; WHI-P151;pyrrolo[2,3-d]-pyrimidines; benzimisazo[4,5-f]isoquinolinonederivatives; AG1801; WP1034; WP1050; WP1015; WP1-1066; WP1129; WP1130;WP1119; WP1026; WP1127; JSI-124; cucurbitacin I; cucurbitacin A;cucurbitacin B; cucurbitacin D; cucurbitacin E; tetrahydro-cucurbitacinI; PD98059 (2′-amino-3′-methoxyflavone); UO126; SL327; olomoucine;5-iodotubercidin; arctigenin; 4-bromo or 4-iodo phenylaminobenzhydroxamic acid derivatives; N3 alkylated benzimidazole derivatives;FR225659; fostriecin; Calyculin A; okadaic acid; cantharidin;TCM-platinum agents containing demethylcantharidin; genistein; MEKinhibitor, and derivatives thereof.

In another embodiment, the agent inhibits the JAK2 signaling pathway,the tyrosine kinase signaling pathway, the Rac/Pak1/Lmk signalingpathway, or the signal transduction activity of CXCR4. In anotherembodiment, the agent is a phosphatase inhibitor.

In another aspect, there is provided a method for identifying compoundsthat inhibit HIV infection, comprising evaluating a compound's abilityto alter the phosphorylation state of a protein of the ADF/cofilinfamily. In one embodiment, the evaluation comprises determining whetherthe compound can inhibit the dephosphorylation of serine-3 residue ofcofilin.

In another aspect, there is provided a composition comprising: (a) aneffective amount of a compound selected from the group consisting of:jasplakinolide; phaloidin; chondramides, chondramide A, B, C, and D;(−)-doliculide; dolastatin-11; dolastatin 3-Nor; majusculamide;dolastatin Hmp; alpha-cyano-3,4-dihydroxy-N-benzylcinnamide (AG490);1,2,3,4,5,6-; JSI-124; benzylidenemalonitriles (“tyrphostins”);WHI-P154; WHI-P151; pyrrolo[2,3-d]-pyrimidines;benzimisazo[4,5-f]isoquinolinone derivatives; AG1801; WP1034; WP1050;WP1015; WP1-1066; WP 1129; WP 1130; WP 1119; WP1026; WP 1127; JSI-124;cucurbitacin I; cucurbitacin A; cucurbitacin B; cucurbitacin D;cucurbitacin E; tetrahydro-cucurbitacin I; PD98059(2′-amino-3′-methoxyflavone); UO126; SL327; olomoucine;5-iodotubercidin; arctigenin; 4-bromo or 4-iodo phenylaminobenzhydroxamic acid derivatives; N3 alkylated benzimidazole derivatives;FR225659; fostriecin; Calyculin A; okadaic acid; cantharidin;TCM-platinum agents containing demethylcantharidin; genistein; andderivatives thereof (b) an effective amount of an anti-retroviral agent;and (c) a pharmaceutically acceptable excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: HIV-1 gp120 mediates depolymerization of cortical actin inresting CD4 T cells. FIG. 1A shows actin polymerization by SDF-1. FIG.1B shows actin depolymerization by gp120. FIG. 1C shows confocalmicroscopy of resting T cells treated with SDF-1 (50 ng/ml), HIV-1 (ngof p24) or gp120 (50 nM), respectively and stained with FITC-phallodin.FIG. 1D shows HIV-1 mediated cortical actin depolymerization. The brightresting T cell (untreated control) and the dime cell (HIV-1 treated)were selected and compared. The relative intensity of F-actin stainingwas measured along arrows (n=1,000 measurements per line) and plotted atthe bottom. FIG. 1E shows cytosolic (non-cortical) F-actin stainingbetween HIV-1 treated and untreated cells detected no difference. Theintensity of cytosolic F-actin staining in randomly selected cells(equal number of infected versus uninfected) was measured along thefolded lines (n=1,000 measurements per line) and shown as the mean±SD onthe right (P>0.117, n=20,000).

FIG. 2: HIV gp120-CXCR4 signaling triggers P.T-sensitive actindepolymerization. FIG. 2A shows actin depolymerization by anti-CD4/CXCR4antibodies conjugated to BD IMag particles. FIG. 2B shows actindepolymerization by anti-CXCR4 but not anti-CD4 BD IMag particles. FIG.2C shows HIV-1 mediated actin depolymerization was dependent onP.T-sensitive signaling. Cells were not treated (left panel) or treatedwith P.T (right panel), followed by HIV-1 infection (ng of p24 per 10⁶cells) and staining with FITC-phallodin for F-actin.

FIG. 3: Inhibition of HIV-1 latent infection by Jas. FIG. 3A graphicallyshows the effects of Jas on HIV-1 latent infection of resting T cells.FIG. 3B shows the effects of Jas on viral entry. FIG. 3C graphicallyshows the effects of Jas on HIV-1 infection of pre-activated T cells.FIG. 3D graphically shows the effects of Jas on HIV-1 infection oftransformed cells. FIG. 3E graphically shows the effects of Jas on HIV-1infection of resting T cells pre-stimulated with anti-CD4/CXCR4 bead (2beads/cell).

FIG. 4: Effects of Lat-A on actin depolymerization and HIV-1 replicationin resting CD4 T cells. FIG. 4A shows dose-dependent actindepolymerization by Lat-A. FIG. 4B shows time-dependent actindepolymerization by Lat-A. FIG. 4C graphically shows enhancement ofHIV-1 latent infection by Lat-A. FIG. 4D graphically showsdose-dependent enhancement of viral replication by Lat-A.

FIG. 5: HIV gp120-CXCR4 signaling triggers P.T-sensitive activation ofcofilin. FIG. 5A provides Western blots of HIV-1 treated cells probedfor P-cofilin, then stripped and reprobed for total cofilin. FIG. 5Bshows activation of cofilin by gp120. FIG. 5C shows activation ofcofilin by anti-CD4/CXCR4 bead (two beads/cell). FIG. 5D showsactivation of cofilin by gp120 promotes its association with actincytoskeleton. F-actin fractions were prepared from gp120 treated restingT cells and immunoblotted for actin (upper band) and cofilin (lowerband). FIG. 5E shows P.T-sensitive activation of cofilin by HIV-1. Cellswere not treated (left panel) or treated (right panel) with P.T theninfected and analyzed as in FIG. 5A. FIG. 5F shows constitutiveactivation of cofilin in transformed cell lines. P-cofilin (upper band)and active cofilin (lower band) was separated by NEPHGE-western blottingand the relative ratio of P-cofilin to active cofilin was indicated atthe bottom. Resting CD4 T cells were used as a control at the right end.FIG. 5G shows F-actin staining of CEM-SS cells infected with HIV-1. FIG.5H shows cofilin activation induced by PHA plus IL-2 and HIV-1 treatmentof resting CD4 T cells.

FIG. 6: Induction of active cofilin promotes viral latent infection ofresting T cells. FIG. 6A shows cofilin specific S3 peptide activatescofilin through competitive inhibition of cofilin phosphorylation byLIMK1. FIG. 6B shows dosage dependent enhancement of viral replicationby S3. FIG. 6C shows viral replication course in cells similarly treatedand infected as in FIG. 6B. FIG. 6D shows that staurosporine inducescofilin activation in resting T cells. Cells were treated withStaurosporine and immunoblotted for P-cofilin and total cofilin. FIG. 6Eshows that staurosporine induces actin depolymerization in resting Tcells. FIG. 6F shows staurosporine induces cofilin activation by directinhibition of LIMK1. FIG. 6G shows enhancement of viral replication byStaurosporine. FIG. 6H shows a model of 120-CXCR4 signaling in mediatingcofilin activation.

FIG. 7: Cytoskeletal actin fractionation of resting CD4 T cells treatedwith gp120.

FIG. 8: Confocal microscopy of CD4 and CXCR4 distribution on resting CD4T cells treated with 120 nM Jas.

FIG. 9: Inhibition of IL-2 secretion by staurosporine.

FIG. 10: Inhibition of T cell activation and viral replication bystaurosporine.

FIG. 11: Activation of Cofilin in HIV positive patients. FIG. 11A is animmunoblot using P-cofilin (Ser3) or cofilin on resting CD4 T cells fromfive HIV patients on HAART therapy and five healthy donors. FIG. 11Bshows the relative ratio of P-cofilin to cofilin. HIV positive donorshave statistically significant lower levels of P-cofilin/cofilin(p=0.001) suggesting higher levels of active cofilin. FIG. 11C shows theabsolute ratios of P-cofilin to active cofilin in three health donorsconfirmed by NEPHEGE western blotting using an anti-cofilin antibody.FIG. 11D shows the absolute ratios of P-cofilin to active cofilin inthree HIV infected donors confirmed by NEPHEGE western blotting using ananti-cofilin antibody.

FIG. 12: Signaling Pathway for Cofilin Activation.

FIG. 13: Screening of HIV inhibitors. JNK inhibitor II inhibitsC-jun-N-terminal Kinase. CA stands for Calyculin A, an inhibitor for thephosphatase PP1a and PP2A. SB90 stands for SB 202190, an inhibitor forp38 Mapkinase. SB74 stands for SB 202474, a negative control for SB 90.FK 506 inhibits PP2B/Calcineurin. Control is cell infected with HIV withno drugs.

FIG. 14: Screening of HIV inhibitors. Genistein is a tyrosine kinaseinhibitor. PD59 is a MAP kinase kinase inhibitor. FK506 is a calcineurininhibitor. Wortmannin is a PI3K inhibitor. LY294002 is a PI3K inhibitor.Control is cell infected with HIV with no drugs.

FIG. 15: Screening of HIV inhibitors. PP2 is a Src family tyrosinekinase inhibitor. PP3 is a negative control for PP2. AG1478 is a EGFRKinase Inhibitor. AG1296 is a PDGF receptor kinase. Control is cellinfected with HIV with no drugs.

DETAILED DESCRIPTION

HIV infection can be detected by measuring signal transduction cascadeevents, such as evaluating phosphorylation levels ofactin-depolymerizing factor (ADF)/cofilin family members. For example,HIV infection of resting T-cells results in cofilin activation bydephosphorylation at serine-3. Thus, monitoring the phosphorylationstate of cofilin can be used to detect HIV in patient samples and tomonitor disease progression. Moreover, inhibitors of phosphorylation ofactin-depolymerizing factor (ADF)/cofilin family members can be used totreat HIV infection.

All technical terms in this description are commonly used inbiochemistry, molecular biology and immunology, respectively, and can beunderstood by those skilled in the field of this invention. Thosetechnical terms can be found in: MOLECULAR CLONING: A LABORATORY MANUAL,3rd ed., vol. 1-3, ed. Sambrook and Russel, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2001; CURRENT PROTOCOLS INMOLECULAR BIOLOGY, ed. Ausubel et al., Greene Publishing Associates andWiley-Interscience, New York, 1988 (with periodic updates); SHORTPROTOCOLS IN MOLECULAR BIOLOGY: A COMPENDIUM OF METHODS FROM CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, 5^(th) ed., vol. 1-2, ed. Ausubel etal., John Wiley & Sons, Inc., 2002; GENOME ANALYSIS: A LABORATORYMANUAL, vol. 1-2, ed. Green et al., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1997; CELLULAR AND MOLECULAR IMMUNOLOGY,4^(th) ed. Abbas et al., WB Saunders, 1994.

DEFINITIONS

Cofilin refers to a protein sequence that reversibly controls actinpolymerization and depolymerization in a pH-sensitive manner. Cofilinhas the ability to bind G- and F-actin in a 1:1 ratio of cofilin toactin. As used herein, cofilin refers to the functionally active cofilinmolecule which is not phosphorylated and which is capable of perturbingthe cell cytoskeleton. Exemplary cofilin polynucleotide and polypeptidesequences are set forth in SEQ ID NO: 1 and 2, respectively.

An “effective amount” is the amount of an agent (or combination ofagents) that is successful in achieving the desired purpose, forexample, treating HIV infection; inhibiting replication of HIV inlatently infected cells; inhibiting HIV infection; etc. The specificdose level and frequency of dosage may vary, and can depend upon avariety of factors, including the activity of the specific activeagents, their metabolic stability and length of action, rate ofexcretion, mode and time of administration, and the age, body weight,general health, gender, diet, severity of the disease, viral load,clinical course of HIV infection, CD4+ T-cell count, of the particularsubject who is undergoing therapy. The combination of agents can besynergistic, i.e., where the joint action of the agents is such that thecombined effect is greater than the algebraic sum of their individualeffects.

The “HIV co-receptor pathway” includes the HIV co-receptors (e.g.,CXCR4, CCR5, etc) that are used for virus entry into cells, anddownstream members of its signaling pathway. Such members include butare not limited to, for example, G-proteins; JAK1, JAK2, JAK3; tyrosinekinases, such as MEK1 and/or MEK2; Rac1/PAK1/LIMK; cofilin;phosphatases, such as PP1, PP2A, hSSH, and/or Chronophin; etc. A memberof co-receptor signaling pathway can also be described as an effector ofco-receptor stimulation, i.e., that is modulated upon co-receptorstimulation by HIV envelope protein.

“Preventing” HIV infection indicates that a subject's susceptibility toHIV infection upon exposure to the virus is reduced or diminished as aresult of the administration of an agent of the present invention. Anyamount of reduced susceptibility (i.e., resistance) is useful, forexample, about 2-fold less, about 5-fold less, about 10-fold less, andany other such improvements can be regarded as preventative.

The term “treat” is used conventionally to mean, for example, themanagement or care of a subject for the purpose of combating,alleviating, reducing, relieving, improving, eliminating, etc., one ormore signs or symptoms associated with HIV infection. Treatment includesdelaying the progression of HIV and its associated symptoms, therebyextending the life expectancy of an infected subject, and/or delaying orreducing the onset of symptoms associated with HIV infection. Treatingcan involve inhibiting, reducing, diminishing, blocking, etc., HIVreplication, especially in latently infected cells; reducing,decreasing, diminishing, etc. HIV viral load in infected subjects;preventing, inhibiting, blocking establishment of HIV latent infection;as well as modulating other events in the life cycle of the HIV virus.

I. Detecting HIV Infection

The actin filament network is involved in a diverse number of cellularprocesses, including control of cell polarization, orientation,intracellular trafficking, membrane fusion, and motility. Proteins ofthe actin-depolymerizing factor (ADF)/cofilin (AC) family dynamicallyregulate these cytoskeletal arrangements by increasing the turnover ofactin. Cofilin activity is regulated by phosphorylation at serine 3which prevents its association with actin, whereas dephosphorylation byphosphatases such as PP1, PP2A or Slingshot (SSH) activates cofilin andpromotes its association with actin and increase actin dynamics.

In eukaryotic cells, the most important physiological function ofcofilin is to sever and depolymerize actin filaments promoting actindynamics. In vitro, ADF/cofilin directly binds to both G- and F-actinand depolymerizes F-actin by changing the twist of actin filaments orstabilizing a preexisting tilted conformation of actin subunits. Invivo, cofilin has been shown to be the primary regulator of corticalactin in yeast, and is among the minimal set of proteins in the actintail required for the motility of the intracellular pathogen Listeria.

In the human immune system, cofilin has been implicated in sustaining Tcell activation. In particular, co-stimulation activates cofilin andpromotes its direct association with F-actin to increase actin dynamics.Cofilin activity is regulated by phosphorylation at serine-3, whichprevents its association with actin, whereas dephosphorylation byphosphatases activates cofilin. Because HIV infection of resting T cellsin vitro results in cofilin activation, the phosphorylation state ofcofilin serine-3 may be used as a diagnostic marker for HIV infection.

A variety of methods for analyzing protein phosphorylation states areknown in the art. For example, proteins can be isolated for westernanalysis using a rabbit anti-cofilin antibody and a rabbitanti-phospho-cofilin (ser3) antibody. In another example, aphosphorylation state can be determined by measuring aphosphorylation-driven conformational change on electron transport. Workby others has demonstrated that helix unfolding impacts the rate ofelectron transport in a dichromomorphic peptide model, resulting in anorder of magnitude difference in electron transport. Fox et al., J. Am.Chem. Soc., 119:5277-5285 (1997). A 10-fold difference, for example, inelectron transport resulting from the addition of the phosphate grouponto the surface of the peptide is likely attributable to aconformational shift within the secondary structure of the peptide.Additionally, phosphorylation may increase the space between atomswithin a peptide, and as a result the entire length of a peptide mayincrease. Cofilin phosphorylation also can be measured by massspectrometry.

II. Treating and/or Preventing HIV

HIV infection can be treated and/or prevented by administering an agentthat modulates the HIV co-receptor signaling pathway stimulated by HIVenvelope protein gp120. In some embodiments, such an agent can inhibitHIV trigger receptor signaling, actin depolymerization; enhance theassembly of actin; stabilize actin filaments; induce polymerization ofmonomeric actin; bind to F-actin or cofilin; and/or inhibit actin andcofilin activity.

The effect of a compound on the cell's cytoskeleton can be determinedroutinely accordingly to any suitable method, including methods commonlyknown in the art. See, for example, Bubb et al., J. Biol. Chem.,276(7):5163-5170 (2000).

Illustrative agents that stabilize or inhibit depolymerization of theactin cytoskeleton include but are not limited to jasplakinolide,phaloidin, chondramides, such as chondramide A, B, C, and D (e.g., Sasseet al., Journal of the National Cancer Institute, 90(20), 1559-1563,1998); (−)-doliculide (e.g., Bai et al., J. Biol. Chem., 277 (35),32165-32171, 2002; Ishiwata et al., J. Org. Chem. 59, 4710-4711, 1994;Ishiwata et al., J. Org. Chem. 59, 4712-4713, 1994); dolastatin-11 andderivatives thereof, such as 3-Nor, Majusculamide, and Hmp derivatives(e.g., Ali et al., Bioorgan. Med. Chem., 13: 3138-4152, 2005). Usefulagents include those which have the same or similar activity tojasplakinolide; inhibit actin depolymerization; enhance the assembly ofpurified actin; stabilize actin filaments in vitro; inducepolymerization of monomeric actin; and/or bind to F-actin.

Cofilin inhibitors include but are not limited to agents that increasethe amount of phosphorylated cofilin (inactive); and/or decrease theamount of functionally active cofilin (i.e., not phosphorylated). Suchagents, for example, promote phosphorylation of cofilin; provide peptidederivatives of cofilin which inhibit cofilin phosphatases, such as hSSHinhibitors. Agents which activate pathways such as Rac1/PAK1,2, Rho/Rockthat lead to activation of LIMK and actin polymerization can inhibitcofilin activity (increase phosphorylation) and inhibit HIV infection ofresting CD4 T cells.

JAK/Stat Signaling Pathway inhibitors can be used to treat and/orprevent HIV infection. Illustrative inhibitors include but are notlimited to JAK1, JAK2, STATS (e.g., STAT1, STAT3 (T-cell sub-type),STAT4, and STAT5), hTid1 (member of the DnaJ family of chaperones),Hsp70, TAT inhibitor oligonucleotides (U.S. Pat. No. 7,002,003);JSI-124, cucurbitacin I, cucurbitacin A, cucurbitacin B, cucurbitacin D,cucurbitacin E, and tetrahydro-cucurbitacin I, and derivatives thereof(e.g., see, U.S. Pub. Appl. No. 2004/0138189),Jalpha-cyano-3,4-dihydroxy-N-benzylcinnamide (AG490);1,2,3,4,5,6-hexabromocyclohexane (Sandberg et al., J Med. Chem. 2005Apr. 7; 48(7):2526-33); JSI-124 (Nefedova et al., Cancer Res. 2005 Oct.15; 65(2):9525-35); benzylidenemalonitriles (“tyrphostins”) (see, also,U.S. Pat. No. 6,433,018; U.S. Pub. Pat. App. No. 2003/0013748);quinazolines (WHI-P154, and WHI-P151); pyrrolo[2,3-d]-pyrimidines;benzimisazo[4,5-f]isoquinolinone derivatives (e.g., U.S. Pat. No.6,852,727); AG1801, WP1034, WP1050, WP1015, WP1-1066, WP 1129, WP1130,WP 1119, WP1026, WP 1127, and derivatives thereof, as well as othercompounds and derivatives thereof (such as WP1002-WP1127), e.g.,disclosed in U.S. Patent Application Publication No. 2005/0277680.

A tyrosine kinase inhibitor can be used to treat and/or prevent HIVinfection. Exemplary inhibitors include but are not limited to MEKinhibitors, such as PD98059 (2′-amino-3′-methoxyflavone), UO126, SL327,olomoucine, 5-iodotubercidin, arctigenin, etc. Other examples of MEKinhibitors are disclosed in U.S. Pub. App. Nos. 20060052608 (e.g.4-bromo or 4-iodo phenylamino benzhydroxamic acid derivatives);20050267012; 20050256123; 20050143438 (N3 alkylated benzimidazolederivatives); 20050130976 (bicyclic inhibitors); 20050059710(diphenylaminoketone); 20050004186; 20040087583(amino-thio-acrylonitriles); 20030092748 (benzenesulfonamidederivatives); 20030045521 (sulfohydroxaminic acids andsulfohydroxamates). EGF receptor and pp 60 v-src kinase inhibitors alsocan be utilized, including, e.g., genistein(4′,5,7-trihydroxyisoflavone) and derivatives thereof (e.g., U.S. Pub.Pat. App. 20030212009), e.g., which have activity on serine andthreonine-dependent protein kinases. Genistein also inhibits kv1.3potassium channels on T-cells (See Teisseyre et al., Membr Biol. 2005May; 205(2):71-9), and has estrogenic activity.

Phosphatase inhibitors also can be used to treat and/or prevent HIVinfection. Phosphatase inhibitors, such as PP1 and PP2A, can inhibitcofilin by dephosphorylation of cofilin. They also may inhibit cofilinactivation through acting on LMK1,2, thereby increasing phospho-LIMK1,2.Other phosphatases examples include, but are not limited to, FR225659(inhibits catalytic subunits of PP1 and PP2A; Hatori et al. J. Antibiot.(Tokyo), 2004 July; 57(7):456-461); fostriecin (e.g., Lewy et al., CurrMed. Chem. 2002 November; 9(22):2005-32); Calyculin A (e.g., Ishihara etal., Biochem Biophys Res Commun. 1989 Mar. 31; 159(3):871-7); okadaicacid; cantharidin; TCM-platinum anticancer agents containingdemethylcantharidin (e.g., To et al., Bioorg Med. Chem. 2004 Sep. 1;12(17):4565-73); and derivatives thereof. Phosphatase assays todetermine inhibitor activity can be carried out routinely, e.g., asdescribed in Soosairajah et al. EMBO J. 9:24(3):473-86 (2005).

Rac signaling pathway inhibitors also can be used to treat and/orprevent HIV infection. Examples include, but are not limited to, RacGTPase inhibitors such as NSC-23766 (Gao et al. PNAS USA, 101:7618-7623, 2004), SCH-51344 (Walsh et al. Oncogene, 5, 2553-2560, 1997);Rho Kinase inhibitors such as HA 1077 (Shirotani et al. J. Pharmacol.Exp. Ther. 259, page 738, 1991),N-(4-Pyridyl)-N′-(2,4,6-trichlorophenyl) urea (Takami et al. Bioorg.Med. Chem. 12, page: 2115, 2004), Y-27632 (Maekawa, et al., Science,285, page: 895, 1999); and PAK inhibitors such as K562a and itsderivatives such as CEP-1347 (Nheu et al. Cancer J., 8, pages: 328-36,2002), and WR-PAK18 (Nheu et al. Cell Cycle, 3, pages: 71-74, 2004).Additionally, genetic approaches also can be used to inhibit the RacSignal Pathway. For example, a dominant-negative Rac1 can directlyinhibit Rac1 (Qiu et al., Nature, 374, pages: 457-459, 1995) andintroducing p57/Kip2 can inhibit LIMK (Yokoo et al. J. Biol. Chem. 278,pages: 52919-52923, 2003).

Table 1 provides a list of inhibitors and there putative sites ofaction.

TABLE 1 Inhibitor Site of Action PD98059 Map Kinase Kinase (MEK)SB203580 p38 Map Kinase U0126 MEK1 and MEK2 Thapsigargin induces releaseof Ca (C₂₄H₃₅N₇ 3HCl 0.5 H₂0) Rac1 Dantrolene blocks Ca release from thesarcoplasmic reticulum SB202190 inhibitor of p38 MapKinase JNK InhibitorII inhibits c-Jun-N-terminal kinase tyrphostin A9 tyrosine kinaseinhibitor, PDGF receptor and Pyk2 C3-exoenzyme Rho inhibitor Y-27632ROCK inhibitor PTP Inhibitor 1 inhibits SHP-1 SB202190 p38 MAP KinaseSB202474 negative control for SB202190 Okadaic Acid PP1alpha, PP2AStaurosporine General Protein Kinase Inhibitor (PKC) Calyculin A PP1alpha, PP2A FK506 calcineurin/PP2B B581 Ras LY-294002 PI3KinaseWortmannin PI3Kinase Vinblastine Microtubule Formation ColchicineMicrotubule Formation Nocodazole Microtubule Formation Taxol MicrotubuleFormation Genistein Protein tyrosine kinase PP2 Src family of tyrosinekinase PP3 Negative control for PP2 Inhibitor AG1478 EGFR kinaseinhibitor AG1296 PDGF receptor kinase JAK2 Inhibitor II JAK2 AG490 EGFRkinase inhibitor/JAK2 4-hydroxyphenacyl Br protein tyrosine phosphatase(PTP) Cytochalasin B actin Cytochalasin D actin Latrunculin A actinSwinholide A actin Misakinolide A actin Tolytoxin actin Mycalolide Bactin Halichondramide actin Aplyronine A actin Pectenotoxin 2 actinPhalloidin actin Jasplakinolide actin Dolastatin 11 actin Hectochlorinactin Doliculide Actin Migrastatin cell migration Motuporamine C cellmigration

Administration of Therapeutic Agents

Therapeutic agents can be administered in any form by any effectiveroute, including but not limited to oral, parenteral, enteral,intraperitoneal, topical, transdermal (e.g., using any standard patch),ophthalmic, nasally, local, non-oral, such as aerosol, spray,inhalation, percutaneous (epidermal), subcutaneous, intravenous,intramuscular, buccal, sublingual, rectal, vaginal, intra-arterial,mucosal, and intrathecal. The agent can be administered alone, or incombination with any ingredient(s), active or inactive.

Any subject can be administered a therapeutic agent, including subjectswho have been exposed to HIV, but have not yet developed HIV infection,as well as subjects who have progressed to one or more of the clinicalsymptoms of HIV infection (e.g., AIDS). In addition to treating and/orpreventing HIV infection in humans, agents can be used to treat otherorganisms (e.g., non-human primates, cats, etc.) infected with HIV, orHIV-related viruses, such as SIV, SHIV, or FIV. Thus, subjects who canbe treated include, e.g., mammals, humans, monkeys, apes, chimpanzees,gorillas, cats, dogs, mice, rats, etc.

A therapeutic agent can be used to treat and/or prevent infection causedby any HIV virus type, including, but is no limited to, HIV-1 (e.g.,clades A, B, C, D, and G, R5 and R5X4 viruses, etc.), HIV-2 (e.g., R5and R5X4 viruses, etc.), simian immunodeficiency virus (SIV),simian/human immunodeficiency virus (SHIV), feline immunodeficiencyvirus (FIV), bovine immunodeficiency virus (BIV) (Wright et al., Vet.Res. Commun., 26:239-50, 2002), HTLV-1, HTLV-2, etc.

In one example, HIV infection can be treated and/or prevented byadministering an effective amount of an agent selected from the groupconsisting of jasplakinolide; phaloidin; chondramides, chondramide A, B,C, and D; (−)-doliculide; dolastatin-11; dolastatin 3-Nor;majusculamide; dolastatin Hmp;alpha-cyano-3,4-dihydroxy-N-benzylcinnamide (AG490); 1,2,3,4,5,6-;JSI-124; benzylidenemalonitriles (“tyrphostins”); WHI-P154; WHI-P151;pyrrolo[2,3-d]-pyrimidines; benzimisazo[4,5-f]isoquinolinonederivatives; AG1801; WP1034; WP1050; WP1015; WP1-1066; WP1129; WP1130;WP1119; WP1026; WP1127; JSI-124; cucurbitacin I; cucurbitacin A;cucurbitacin B; cucurbitacin D; cucurbitacin E; tetrahydro-cucurbitacinI; PD98059 (2′-amino-3′-methoxyflavone); UO126; SL327; olomoucine;5-iodotubercidin; arctigenin; 4-bromo or 4-iodo phenylaminobenzhydroxamic acid derivatives; N3 alkylated benzimidazole derivatives;FR225659; fostriecin; Calyculin A; okadaic acid; cantharidin; andTCM-platinum agents containing demethylcantharidin.

Pharmaceutical Combinations

Therapeutic agents also can be combined with other agents, especiallyagents which are utilized to treat HIV. Examples of drugs which can becombined with an agent of the present invention include but are notlimited to protease inhibitors, reverse transcriptase inhibitors(includes nucleoside/nucleotide drugs and non-nucleoside inhibitors),integrase inhibitors, attachment inhibitors, chemokine receptorinhibitors, RNAase H inhibitors, entry inhibitors; assembly and buddinginhibitors; etc. Classes of HIV drugs include but are not limited toattachment inhibitors (TNX-355, BMS-488043), CCR5 coreceptor antagonists(SCH-D, UK-427857, GW 873140) and a maturation inhibitor (PA-457). See,McNicholl and McNicholl, Curr Pharm Des. 12(9):1091-103.

Examples of HIV drugs which can be combined with agents include, but arenot limited to, abacavir, saquinavir, ritonavir, indinavir, nelfinavir,amprenavir, lopinavir, atazanavir, fosamprenavir, tipranavir,enfuvirtide, tenofovir, entricitabine, AZT, ddI, ddC, ddT, 3TC, d4T,ZDV, nevirapine, delavirdine, etc. In addition, drugs also include,e.g., BMS-48804; UK427,857; TAK-652; GW 871340; CMPD 167; AMD3100;Amdoxovir; TMC278; BILR 355BS; Capravirine; KMMP05; L-870810; FZ41;Tipranavir; TMC114; UIC-020301; GW640385; AG-001859; PA-457; etc. Agentsof the present invention also can be combined with vaccines and otherpreventative measures. See, e.g., U.S. Pat. No. 6,962,900

Suitable excipients for use in a pharmaceutical composition include, butare not limited to, a lubricant, glidant, diluent, binder, disintegrant,carrier, colorant, or coating material. Examples of pharmaceuticallyacceptable excipients include, but are not limited to, lactose, sugar,corn starch, modified corn starch, mannitol, sorbitol, silicon dioxide,and microcrystalline cellulose.

Anti-retroviral agents attempt to treat HIV infections by inhibitingreplication of the HIV virus by blocking the reverse transcriptase or byblocking the HIV protease. Three categories of anti-retroviral agents inclinical use are nucleoside analogs (such as AZT), protease inhibitors(such as nelfinavir), and the recently introduced non-nucleoside reversetranscriptase inhibitors (NNI) such as nevirapine. Any anti-retroviralagent may be used in a pharmaceutical composition.

Vaccines

HIV vaccines for treating and/or preventing HIV infection can comprise agp120 polypeptide in which the chemokine signaling domain is deleted ormutated to eliminate or reduce its ability to stimulate the cellsignaling activity of its cognate chemokine receptor. Such a gp120polypeptide can contain one or more of the following mutations: V1/V2D133; V1/V2 K135; V1/V2 D137; V1/V2 S143; V1/V2 R146; V1/V2 E153 V1/V2R166; V1/V2 K168; V1/V2 Q170; V3 R296; V3 S304; V3 R309; V3 R313; V3F315; V3 V316; V3 Q326; V3 R325; β19 I418; β19 K419; β19 Q420; deletionsinvolving V1/V2, V3, and/or β19 (including complete or partialdeletions. See, e.g., Kwong et al., Nature, 393:649-659, 1998.Polypeptides can be produced routinely using suitable recombinanttechnologies.

III. Identifying Compounds Inhibiting HIV Infection

Compounds inhibiting HIV infection can be identified by contacting aT-cell with a test agent, contacting the T-cell with infectious HIVunder conditions effective for said HIV to latently infect said T-cell,activating the latently infected T-cell to produce an activated T-cell,and determining the amount of HIV produced from the activated T-cell,wherein the amount indicates whether the agent inhibits HIV infection;determining the amount of HIV produced from latently infected andactivated T-cells which have been pre-treated with a test agent. Thepresence of HIV particles can be determined routinely, for example, bymeasuring polypeptides or RNA, such as p24, reverse transcriptaseactivity; viral RNA, etc. Such measurements can be made usingimmunoassays, PCR, etc.

The cell cytoskeleton can be used as an indicator of test agentefficiency. For example, phalloidin can be utilized to investigate thedistribution of F-actin in cells by labeling phalloidin with fluorescentanalogs and using them to stain actin filaments. A high-resolutiontechnique can be used to detect F-actin at the light and electronmicroscopic levels with phalloidin conjugated to the fluorophore eosinwhich acts as the fluorescent tag. See, Capani et al., J HistochemCytochem. 2001 November; 49(11):1351-61. In this method, known asfluorescence photo-oxidation, fluorescent molecules can be utilized todrive the oxidation of diaminobenzidine (DAB) to create a reactionproduct that can be rendered electron dense and detectable by light andelectron microscopy.

The state of actin cytoskeleton also can be assayed usingFITC-phalloidin, or other detectable phalloidin conjugates. Briefly,cells (such as primary T-cells) can be contacted with a suitablepermeabilizing agent (e.g., triton). The cells can be washed and thencontacted with FITC-phalloidin under conditions suitable for thephalloidin to bind to the actin filaments. The labeled cells can befixed in paraformaldehye, or other suitable fixative, and thenvisualized, e.g., using microscopy or flow cytometry. Pre-treatment ofprimary T-cells with HIV (or gp120 alone) results in less intensestaining, indicating the actin network has been reduced, for example, bydepolymerization, etc.

Specific examples are presented below of methods for detecting andtreating and/or preventing HIV. They are meant to illustrate and not tolimit the present invention.

Example 1 Isolation of Resting CD4 T Cells from Peripheral Blood

Peripheral blood mononuclear cells (PBMC) were obtained from healthydonors at the Student Health Center, George Mason University (GMU),Fairfax, Va. All protocols involving human subjects were reviewed andapproved by the GMU IRB. Resting CD4 T cells were purified by two roundsof negative selection as previously described in Wu and Marsh, Science293(5534), 1503-6 (2001). Purified cells were cultured in RPMI 1604medium supplemented with 10% heat-inactivated fetal bovine serum(Invitrogen, Carlsbad, Calif.), penicillin (50 U/ml) and streptomycin(50 μg/ml). Cells were rested overnight before infection or treatment.

Example 2 Virus Preparation and Infection of Resting CD4 T Cells

Virus stocks of the HIV-1_(NL4-3) (Adachi et al. J Virol 59(2), 284-91(1986)) were prepared by transfection of HeLa cells with cloned proviralDNA as described in Wu and Marsh (2001). Supernatant was harvested at 48hours, and filtered through a 0.45 μm nitrocellulose membrane. Virustiter (TCID50) was measured by infection of a Rev-CEM indicator cellline which was constructed through stable integration of a Rev-dependentGFP expression lentiviral vector into CEM-SS. This Rev-CEM cell line hasno background GPF expression and gives titers close to those by usingPBMC. For infection of resting CD4 T cells, viruses were first titratedto determine minimal levels detectable by p24 ELISA in replicationassays described below. Unless specified, for most replication assays,10^(3.5) to 10^(4.5)TCID50 units of HIV-1 were used to infect 10⁶ cells.For infection procedure, CD4 T cells were incubated with the virus for 2hours, then washed twice with medium to remove unbound virus. Infectedcells were resuspended into fresh RPMI 1604 medium supplemented with 10%heat-inactivated fetal bovine serum, penicillin (50 U/ml) andstreptomycin (50 μg/ml) at a density of 10⁶ per ml and incubated for 5days without stimulation. Cells were activated at day 5 withanti-CD3/CD28 magnetic beads at 4 beads per cells. For viral replicationassay, 10% of infected cells were taken at 1, 3, 5, 6, 7, 8, 9 days postinfection. Cells were pelleted and the supernatant was saved for p24ELISA. Levels of p24 in the supernatant were measured by using CoulterHIV-1 p24 Assay Kit (Beckman Coulter, Inc. Miami, Fla.). Plates werekinetically read using ELx808 automatic microplate reader (Bio-TekInstruments, Inc. Winooski, Vt.) at 630 nm.

Example 3 Pre-Treatment of Resting CD4 T Cells with Inhibitors

Resting CD4 T cells were treated with pertussis toxin (Sigma, St. Louis,Mo.) at a final concentration of 100 ng/ml for 2 hours, then infectedwith HIV-1. Following infection, cells were washed 2 to 3 times toremove cell-free virus and the inhibitor. Cells were treated withJasplakinolide (Molecular Probes, Eugene, Oreg.) for 1 to 2 hours orLatrunculin A (Biomol, Plymouth Meeting, Pa.) for 5 minutes at variousconcentrations, washed twice with medium, then infected with HIV-1.Following infection, cells were washed twice to remove cell free virus.Cells were treated with 200 nM of Staurosporine (Biomol, PlymouthMeeting, Pa.) for 2 hours, then infected with HIV-1. Followinginfection, cells were washed twice to remove cell-free virus and theinhibitor.

To detect effects of Staurosporine on cofilin activation and actindepolymerization, cells were treated with 200 nM of Staurosporine forvarious times, then pelleted and lysed in NuPAGE LDS Sample Buffer(Invitrogen, Carlsbad, Calif.) for western blotting, or directly fixedand permeabilized for F-actin staining For activation of resting CD4 Tcells with PHA (3 μg/ml) (Sigma, St. Louis, Mo.) plus IL-2 (100 U/ml)(Roche Applied Science, Indianapolis, Ind.), cells were cultured in thepresence of these agents for 1 day.

Example 4 Pre-treatment of Resting CD4 T Cells with Synthetic Peptide

Synthetic peptide S3 (MASGVAVSDGVIKVFN; SEQ ID NO: 3) was derived fromthe N-terminal 16 amino acids of human cofilin and control peptide Q104(WAPESAPLQSQM; SEQ ID NO: 4) was derived from human cofilin residues 104to 115 with lysine 112 and 114 mutated to glutamine. Both peptides weresynthesized by Celtek Peptides (Nashville, Tenn.) and conjugated to apenetratin peptide (RQIKIWFQNRRMKWKK; SEQ ID NO: 5) for intracellulardelivery. Resting CD4 T cells were treated with S3 or Q104 for 1 to 2hours, then infected with HIV-1. Following infection, cells were washed2 to 3 times to remove cell-free virus and the peptides.

Example 5 Conjugation of Antibodies to Magnetic Beads and Stimulation ofResting CD4 T cells

Monoclonal antibodies again Human CD3 (clone UCHT1), CD28 (cloneCD28.2), CD4 (clone PRA-T4) and CXCR4 (clone 12G5) were from BDPharmingen (BD Biosciences, San Diego, Calif.). The anti-CD4, CXCR4antibodies were selected for their shared epitopes with gp120. The CD4antibody, clone RPA-T4, binds to the D1 domain of the CD4 antigen and iscapable of blocking gp120 binding to CD4 (Dalgleish et al., Nature312(5996), 763-7 (1984)), whereas the anti-CXCR4 antibody, clone 12G5,interacts with the CXCR4 extracellular loop 1 and 2 which partiallyoverlap domains for the HIV-1 coreceptor function. Lu et al., Proc NatlAcad Sci USA 94(12), 6426-31 (1997). The 12G5 antibody also has beenshown to block HIV-1 mediated cell fusion (Hesselgesser et al., JImmunol 160(2), 877-83 (1998)) and CD4-independent HIV-2 infection.Endres et al., Cell 87: 745-756 (1996). For conjugation, 10 μg ofantibodies were conjugated with 4×10⁸ Dynal beads (Invitrogen, Carlsbad,Calif.) for 30 min at room temperature. Free antibodies were washed awaywith PBS-0.5% BSA and magnetic beads were resuspended in 1 ml ofPBS-0.5% BSA.

For stimulation of resting CD4 T cells, antibody conjugated beads werewashed twice, then added to cell culture and rocked for 5 min.Conjugation of anti-CD4, CXCR4 antibodies with Streptavidin-labeled BDIMag particles (BD Biosciences, San Diego, Calif.) was carried out byusing 25 μl of washed particles and 125 μl of biotin-labeled anti-CD4,CXCR4 antibodies (BD Biosciences, San Diego, Calif.). The mixture wereincubated for 30 min at room temperature with gentle shaking, and washedthree times and resuspended into 250 μl of PBS-0.1% BSA. Resting CD4 Tcells were treated at 10 μl particles per 10⁶ cells.

Example 6 FITC-Phallodin Staining of F-actin and Flow Cytometry

Cells were stimulated with HIV-1 or gp120 IIIB (Microbix Biosystems Inc.Toronto, Ontario). F-actin staining using FITC-labeled phalloidin(Sigma, St. Louis, Mo.) was carried out according to the manufacture'srecommendation with minor modifications. Briefly, each staining wascarried out by using 10⁶ cells. Cells were pelleted, fixed andpermeabilized with cytoperm/cytofix (BD biosciences, San Diego, Calif.)for 20 min on ice, followed by stained with 5 μl of 0.3 uM FITC-labeledphalloidin for 30 min at 4° C. After wash, cells were resuspended in 1%paraformaldehyde and analyzed on FACSCalibur (BD Biosciences, San Jose,Calif.)

Example 7 Staining of LAF-1 Activation on Resting CD4 T Cells

Half million CD4 T cells in 500 μl culture medium were treated with 5 μlof human IgG (Jackson Immuno Research, West Grove, Pa.) to blocknon-specific staining Cells were stained with 10 μl (50 μg/ml) of humanICAM-1 Fc chimera (R&D System, Minneapolis, Minn.) for 20 min at 4° C.Cells were washed and blocked by 5 μl of mouse IgG (JacksonImmunoResearch, West Grove, Pa.), followed by the addition of 10 μl ofFITC labeled mouse anti-human Fc (Jackson ImmunoResearch, West Grove,Pa.), and incubated for 20 min at 4° C. Cells were finally washed andresuspended in 500 μl of 1% paraformaldehyde for flow cytometry.

Example 8 Confocal Microscopy

Stained cells were imaged using a Zeiss Laser Scanning Microscope(Thornwood, N.Y.), LSM 510 META, with a 40 NA 1.3 or 60 NA 1.4 oil DicPlan-Neofluar objective. Samples were excited with two laser lines, 488nm for GFP and 543 nm for Alexa 594. Images were simultaneously recordedin three channels: channel one: fluorescent emissions from 505 to 530 nmfor GFP (green); channel two: emissions from 580 to 650 nm for Alexa 594(red); channel three: DIC. Images were processed and analyzed by the LSM510 META software.

FIG. 8 illustrates confocal microscopy of CD4 and CXCR4 distribution onresting CD4 T cells treated with 120 nM Jas. Resting CD4 T cells weretreated with 120 nM of Jas for 2 hours, then fixed and stained with aFITC-labeled anti-human CD4 monoclonal antibody and a biotin-labeledanti-human CXCR4 monoclonal antibody followed by Alexa Fluor-594 labeledstreptavidin (right panel). Untreated resting CD4 T cells were used as acontrol (left panel).

Example 9 PCR Amplification

To remove plasmid DNA contamination, viral stock was treated withBenzonase (Novagen, Madison, Wis.) (250 U/ml) at 37° C. for 15 minbefore infection. Total cellular DNA were purified using SV total RNAisolation kit as recommended by the manufacture (Promega, Madison,Wis.). For detection of viral late reverse transcription products byPCR, forward primer HIV/Tat-Rev-5′ (5′ GGTTAGACCAGATCTGAGCCTG 3′; SEQ IDNO: 6) and reverse primer LTR-gag2 (5′ TTAATACCGACGCTCTCGCACC 3′; SEQ IDNO: 7) were used. PCR was carried out in 1× Ambion PCR buffer, 125 μMdNTP, 50 pmol each primer, 1U SuperTaq Plus (Ambion Inc. Austin, Tex.)with 30 cycles at 94° C. for 20 seconds, 68° C. for 60 seconds. Forrelative quantification, the cellular-actin pseudogene were co-amplifiedusing QuantumRNA-actin Internal Standards (Ambion Inc. Austin, Tex.),with a ratio from 5/5 to 1/9 for actin primer/competitor.

Example 10 Cofilin and Phospho-Cofilin Western Blot

Briefly, 10⁶ of CD4 T cells were lysed in NuPAGE LDS Sample Buffer(Invitrogen, Carlsbad, Calif.) followed by sonication. Samples wereheated at 70° C. for 10 minutes before loading, then separated bySDS-PAGE and transferred onto nitrocellulose membranes (Invitrogen,Carlsbad, Calif.). The membranes were washed in TBS for 5 minutes andthen blocked for 30 minutes at room temperature with Starting Blockblocking buffer (Pierce, Rockford, Ill.). The blots were incubated witheither a rabbit anti-cofilin antibody (1:1000 dilution) (Cell Signaling,Danvers, Mass.) or a rabbit anti-phospho-cofilin (ser3) antibody (1:1000dilution) (Cell Signaling, Danvers, Mass.) diluted in 5% BSA-TBST androcked at room temperature for 1 hour. The blots were washed three timesfor 15 minutes each and then incubated with a goat anti-rabbithorseradish peroxidase-conjugated antibodies (KPL, Inc. Gaithersburg,Md.) diluted in 2.5% skim milk-TBST (1:1000) for 1 hour. The blots werewashed again three times for 15 minutes each and then developed withSuperSignal west femto maximum sensitivity substrate (Pierce, Rockford,Ill.). Images were captured with a CCD camera (Fluor Chem 9900 ImagingSystems) (Alpha Innotech, San Leandro, Calif.) and analyzed andprocessed by NIH-Image Version 163.

Example 11 G-actin/F-actin Fractionation and F-actin/cofilinCosedimentation Assays

G-actin/F-actin cellular fractions were prepared using theG-actin/F-actin in vivo assay Kit (Cytoskeleton, Inc, Denver, Co).Briefly, 2 million resting CD4 T cells per sample were treated with 500pM gp120 from 5 minutes to 1 hour. The cells were harvested bycentrifugation at 2,000 g for 1 minute at 37° C. and then resuspended in1.5 ml lysis buffer (50 mM PIPES pH6.9, 50 mM KCl, 5 mM MgCl2, 5 mMEGTA, 5% (v/v) Glycerol, 0.1% Nonidet P40, 0.1% Triton X-100, 0.1% Tween20, 0.1% 2-mercapto-ethanol, 0.001% Antifoam C and 4 uM Tosyl argininemethyl ester, 15 uM Leupeptin, 10 uM Pepstatin A, 10 mM Benzamidine and1 uM ATP). The lysates were homogenized with a 200 μl fine orificepipette and then placed at 37° C. for 10 minutes. The lysates were thencentrifuged at 420 g for 5 minutes to remove cell debris and thesupernatant was collected and centrifuged at 100,000 g at 37° C. for 1hour. Following centrifugation, the supernatant containing G-actin wassaved and the pellet containing F-actin was resuspended in F-actindepolymerizing solution (10 μM cytochalasin D) and incubated on ice for1 hour with occasional pipetting. Equal volumes of the supernatant andthe pellet factions were used for SDS PAGE and western blotting foractin. For the F-actin/cofilin cosedimentation assay, the F-actin pelletwas directly resuspended in 1×LDS sample buffer for SDS-PAGE and westernblotting for cofilin and actin.

Example 12 NEPHGE and Western Blot for Cofilin

One-dimensional NEPHGE was performed as previously described in Nebl etal. Cell Signal 16(2), 235-43 (2004)). Briefly, resting T cells werelysed in TKM buffer (50 mM Tris pH 7.6, 25 mM KCL, 5 mM MgCL₂, 1 mMNa-Vandate, 5 mM NaF, 20 ug/ml Leupeptin, 20 ug/ml Aprotinin, 0.3 uMokadaic acid containing 0.5-1% NP-40) and sedimented at 20,800 g for 10minutes at 4° C. Postnuclear fraction was collected and used for thedetection of cofilin. NEPHGE were performed with 6%-focusing slab gelwith 5% Ampholines pH 3 to 10 (Invitrogen, Carlsbad, Calif.). The gelswere run for 1 hour at 100V, 2 hours at 250V and 2 hours at 300V, thentransferred onto nitrocellulose membrane (Invitrogen, Carlsbad, Calif.)and incubated with 1:1000 dilution of a rabbit anti-cofilin antibody(Cell Signaling, Danvers, Mass.), followed by incubation with 1:1000dilution of a goat anti-rabbit antibody conjugated with horseradishpreoxidase (KPL, Gaithersburg, Md.). Signals were acquired by usingSuperSignal west femto maximum sensitivity substrate (Pierce, Rockford,Ill.) and a CCD camera (Fluor Chem 9900 Imaging Systems) (AlphaInnotech, San Leandro, Calif.). Images were analyzed and quantifiedusing NIH-Image Version 163 as suggested by the software developer.

Example 13 In vitro LIMK Kinase Assay

LIMK1 Kinase assays were performed using purified LIMK1 and GST-taggedrecombinant human cofilin (Upstate Biotechnologies, Lake Placid, N.Y.)according to the manufacture's recommendation with minor modifications.Briefly, 18 μg of recombinant cofilin was incubated in 1× Kinasereaction buffer (800 nM MOPS-NaOH, pH7.0, 200 μM EDTA) in the presenceor absence of Staurosporine (200 nM) or the S3, Q104 peptides. LIMK1 wasserially diluted in dilution buffer (20 mM MOPS-NaOH pH 7.0, 1 mM EDTA,0.01% Brij-35, 5% glycerol, 0.1% 2-ME, 1 mg/ml BSA), then added into thereaction along with the ATP buffer (10 mM Magnesium Acetate, 100 μMATP). The reaction was incubated for 15 minutes at 30° C. with constantagitation. The reaction was stopped by adding 25% (V/V) of 4×LDS samplebuffer for SDS-PAGE and heated for 10 minutes at 70° C. Cofilinphosphorylation was analyzed by SDS-PAGE and western blotting using arabbi anti-phospho-cofilin (ser3) antibody (Cell Signaling, Danvers,Mass.) as described in Example 10.

Example 14 IL-2 ELISA

IL-2 release into cell culture supernatant was detected by a human IL-2ELISA development kit (PeproTech, Rocky Hill, N.J.) according to themanufacture's instruction. Briefly, each well of a plate was coated with100 μl of capture antibody (1 μg/ml) and incubated overnight at roomtemperature, then washed and blocked with 300 μl of blocking solutionfor one hour at room temperature. Samples in plates were incubated for 1hour at 37° C., then washed and incubated with 100 μl of detectionantibody (0.5 μg/ml) for 1 hour at 37° C. Plates were washed andincubated with 100 μl of the avidin-peroxidase conjugate (1:2000dilution) for 30 minutes at room temperature followed by washing andincubation with 100 μl of Tetramethylbenzidine (TMB) substrate buffer.Plates were kinetically read using ELx808 automatic microplate reader(Bio-Tek Instruments, Inc. Winooski, Vt.) at 630 nm.

Example 15 gp120 Induces Cytoskeletal Rearrangement

To determine the nature of the cytoskeletal rearrangement induced bygp120, filamentous actin (F-actin) change was compared followingtreating resting T cells with SDF-1, the natural ligand for CXCR4, orwith gp120. In SDF-1 treated cells rapid actin polymerization (FIG. 1A)was observed with a characteristic of highly polarized cortical actin(12) (FIG. 1C). Conversely, in gp120 treated cells, actindepolymerization started at 5 mm and became more pronounced at 1 to 3hours (FIG. 1B). Depolymerization was consistently observed in multipledonors and across a range of gp120 concentrations below 50 nM.

As shown in FIG. 1, HIV-1 gp120 mediates depolymerization of corticalactin in resting CD4 T cells. FIG. 1B shows actin depolymerization bygp120. Cells were treated with gp120IIIB (50 nM) and stained withFITC-phalloidin and analyzed by flow cytometer. Shown is histogram. FIG.1A shows actin polymerization by SDF-1. Cells were treated with SDF-1(50 ng/ml) and stained with FITC-phalloidin. FIG. 1C shows confocalmicroscopy of resting T cells treated with SDF-1 (50 ng/ml), HIV-1 (ngof p24) or gp120 (50 nM), respectively and stained with FITC-phallodin.Images were acquired in identical condition. Red arrows indicatelocalized actin polymerization induced by SDF-1 or gp120. FIG. 1D showsHIV-1 mediated cortical actin depolymerization. The bright resting Tcell (untreated control) and the dime cell (HIV-1 treated) were selectedand compared. The relative intensity of F-actin staining was measuredalong the red arrow lines (n=1,000 measurements per line) and plotted atthe bottom. The major difference detected was in the cortical actinregion. FIG. 1E shows cytosolic (non-cortical) F-actin staining betweenHIV-1 treated and untreated cells detected no difference. The intensityof cytosolic F-actin staining in randomly selected cells (equal numberof infected versus uninfected) was measured along the folded red lines(n=1,000 measurements per line) and shown as the mean±SD on the right(P>0.117, n=20,000).

These data suggest distinct differences between SDF-1 and gp120 instimulating cellular responses, despite their shared similarities inCXCR4 binding. K. Balabanian et al., J Immunol 173, 7150 (2004). WhileSDF-1 triggers physiological responses, gp120 likely mediates aberrantsignaling with potential pathogenic consequences. Additionally, unlikeSDF-1, gp120 engages both CXCR4 and CD4.

Example 16 gp120 Depolymerizes Actin

To further confirm actin depolymerization by gp120, cellularfractionation was performed to measure changes in the ratio of F-actinto globular actin monomer (G-actin) in response to gp120 treatment.There was a significant increase in the G/F actin ratio following gp120treatment, consistent with the data from FITC-phalloidin staining (FIG.7). Finally, confocal microscopy revealed that the actindepolymerization in HIV treated cells mainly occurred in the corticalactin region in resting CD4 T cells (FIG. 1D). No significant differencewas observed in the intracellular F-actin staining beyond the corticalactin between treated and untreated cells (FIG. 1E). Thus, HIV-1 gp120largely depolymerizes the cortical actin in resting T cells.

Example 17 CD4 and CXCR4 in gp120 Mediated Actin Depolymerization

To define specific roles of CD4 and CXCR4 in gp120 mediated actindepolymerization, resting T cells were stimulated with magneticparticles coated with antibodies against both receptors. FIG. 2.illustrates that HIV gp120-CXCR4 signaling triggers P.T-sensitive actindepolymerization. (A) Actin depolymerization by anti-CD4/CXCR4antibodies conjugated to BD IMag particles. (B) Actin depolymerizationby anti-CXCR4 but not anti-CD4 BD IMag particles. (C) HIV-1 mediatedactin depolymerization was dependent on P.T-sensitive signaling. Cellswere not treated (left panel) or treated with P.T (right panel),followed by HIV-1 infection (ng of p24 per 10⁶ cells) and staining withFITC-phallodin for F-actin.

FIG. 2A shows actin depolymerization following antibody stimulation.When cells were stimulated with either anti-CD4 or anti-CXCR4 particles,actin depolymerization occurred only with CXCR4 stimulation, while CD4stimulation slightly enhanced actin polymerization (FIG. 2B). Thus,CXCR4 is sufficient to mediate actin depolymerization. Notably, theability of these antibodies to induce actin depolymerization correlateswith their ability to enhance viral replication.

Example 18 Gai-mediated Actin Depolymerization

Actin polymerization induced by SDF-1 is regulated through binding toCXCR4 and subsequent activation of the G proteins (M. P. Crump et al.,Embo J 16, 6996 (1997); X. Huang et al., Biophys J 84, 171 (2003)),particularly pertussis toxin (P.T) sensitive Gai (Y. Sotsios, et al.,Journal of Immunology 163, 5954 (1999)). To determine whether thedepolymerization induced by gp120 is likewise mediated through Gai,resting T cells were treated with P.T and found that P.T completelyinhibited viral induced actin depolymerization (FIG. 2C), suggestingthat the depolymerization also is mediated through CXCR4 and the Gaiprotein. While the same receptor mediates both actin polymerization anddepolymerization, SDF-1 has been reported to both attract and repel Tcells depending on dosages. M. C. Poznansky et al., Nat Med 6, 543(2000). The fact that P T inhibited viral replication as well as actindepolymerization suggests that the depolymerization could be one of theessential functions of gp120-CXCR4 signaling in priming viral latentinfection of resting T cells.

Example 19 Effect of Jas on T Cell Activity

To test whether cortical actin in resting T cells is a barrier thatneeds to be depolymerized, a F-actin stabilizing agent's, jasplakinolid(Jas), ability to interfere with HIV envelope-induced actindepolymerization was evaluated. Similar to phalloidin, Jas binds toF-actin irreversibly and stabilizes actin filaments M. R. Bubb, et al.,J Biol Chem 275, 5163 (2000). Given that the actin cytoskeleton also isinvolved in T cell activation, Jas may affect HIV replication indirectlyby affecting T cell activity. For instance, the actin cytoskeleton isknown to be the driving force for receptor clustering and the formationof the supramolecular activation cluster (SMAC) during T cellactivation. C. Wulfing and Davis, M. M., Science 282, 2266 (1998). Thisprocess involves the actin-dependent activation of LFA-1 (Y. van Kooyk,et al., J Biol Chem 274, 26869 (1999); M. L. Dustin and T. A. Springer,Nature 341, 619 (1989)), which is required for sustained signaling toreach full T cell activation. C. Wulfing, et al., Proc Natl Acad Sci USA95, 6302 (1998).

Thus, the effect of Jas on T cell activity was determined. FIG. 3Aillustrates inhibition of HIV-1 latent infection by Jas. Effects of Jason LFA-1 activation also were tested. Jas treated or untreated resting Tcells were activated by anti-CD3/CD28 bead and stained with humanICAM-1/Fc chimera and analyzed by flow cytometer. The effects of Jas onIL-2 secretion also were measured. FIG. 3A shows the effects of Jas onHIV-1 latent infection of resting T cells. Cells were treated with Jasfor 2 hours, washed, infected with HIV-1 for 5 days, then activated byanti-CD3/CD28 bead. FIG. 3B shows the effects of Jas on viral entry.Following infection, cellular DNA was PCR amplified for viral late DNAand β-actin pseudogene. The effects of Jas on HIV-1 infection ofpre-activated T cells are shown in FIG. 3C. Cells were pre-activatedwith PHA plus IL-2, then treated and infected as in FIG. 3A. FIG. 3Dshows the effects of Jas on HIV-1 infection of transformed cells. CEM-SScell were treated and infected as in FIG. 3A. Effects of Jas on HIV-1infection of resting T cells pre-stimulated with anti-CD4/CXCR4 bead (2beads/cell) are shown in FIG. 3E. Pre-stimulated cells were treated andinfected as in FIG. 3A.

Staining of resting T cells with ICAM-1, the ligand for active LFA-1,confirmed that T cell activation induced LFA-1 activation. However,treatment of T cells with 3 μM Jas greatly inhibited LFA-1 activation.Therefore, Jas was titrated to lower dosages. It was found that at 120nM and below, Jas has no detectable inhibition on LFA-1 activation.Using secretion of IL-2 as a second indicator for T cell activity, itwas confirmed that at 120 nM and below, Jas had no significant effect onIL-2 secretion although at 600 nM and above it inhibited IL-2 expressionfollowing T cell activation. Effects of Jas on T cell activity werefurther tested by cell cycle analysis. At 3 μM, Jas arrested cells in Sphase, whereas at 120 nM, no cell cycle arrest was observed (data notshown).

From these results, it was determined that a Jas concentration below 120nM could be used to test its effects on HIV replication (FIG. 3A). At120 nM, Jas retains the characteristics of irreversible binding toF-actin and significantly inhibited the subsequent competitive bindingof phalloidin (data not shown). Given its minimal impact on T cellactivity at lower dosages, Jas was used to treat resting T cells for 2hours, then cells were infected. Complete inhibition of HIV replicationwas observed at 600 and 120 nM and partial inhibition at 24 nM (FIG.3A). Jas inhibition was observed in multiple donors (data not shown) andthe inhibition was not due to inhibition of gp120 mediated CD4/CXCR4receptor clustering which has been suggested to be actin dependent. S.Iyengar, et al., J Virol 72, 5251 (1998). Confocal microscopy did notreveal any significant difference in CD4/CXCR4 distribution on thesurface between 120 nM Jas treated and untreated cells (FIG. 8). Neitherwas the inhibition due to effects on viral-cell fusion which also hasbeen suggested to be actin dependent. S. E. Pontow, et al., Journal ofVirology 78, 7138 (2004). Quantitative PCR amplification ofintracellular viral DNA did not detect any difference between 120 nM Jastreated and untreated cells, suggesting that the inhibition was not atthe entry level (FIG. 3B).

These results show that actin rearrangement mediated by the HIV envelopeis critical for a post entry process, such as uncoating or intracellularmigration. Contrary to resting CD4 T cells, similar Jas treatment oftransformed CEM-SS or pre-activated T cells had no inhibition on HIVreplication (FIGS. 3, C and D). It is likely that at 120 nM, Jas has noinhibition on actin remodeling mediated by cell cycle. At a higherdosage (3 μM), however, when cell cycle was arrested, Jas did inhibitHIV replication in CEM-SS (data not shown). These data also confirm thatat 120 nM, Jas does not affect viral entry or reverse transcription(FIG. 3B). Also, pre-stimulation of resting T cells with CD4/CXCR4 alsocompletely abolished the inhibition by 120 nM Jas, suggesting that theactin cytoskeleton in resting T cells may no longer be a barrier oncereorganized by the CD4/CXCR4 pre-stimulation (FIG. 3E).

Example 20 Effect of Lat-A on T Cells

To further confirm that the cortical actin cytoskeleton constitutes arestriction in resting T cells, another actin inhibitor, Latraculin A(Lat-A) was investigated, which has been shown to specifically induceactin depolymerization without affecting microtubules. I. Spector, etal., Science 219, 493 (1983); M. Coue, et al., FEBS Lett 213, 316(1987); and K. R. Ayscough et al., J Cell Biol 137, 399 (1997). UnlikeJas, Lat-A does not directly interact with F-actin to promote actindisassembly (Ayscough (1997)), rather it binds to G-actin reversibly andinhibits its assembly into filamentous actin. Spector (1983); Ayscough(1997).

It was speculated that artificial induction of actin depolymerization byLat-A may enhance HIV replication if cytoskeletal actin serves as abarrier. To test this, Lat-A was titrated at various dosages from 2.5 μMto 2.5 nM, and actin depolymerization was examined in resting CD4 Tcells.

FIG. 4. shows the effects of Lat-A on actin depolymerization and HIV-1replication in resting CD4 T cells. FIG. 4A shows dose-dependent actindepolymerization by Lat-A. Cells were treated with Lat-A for 5 min, thenstained for F-actin and analyzed by flow cytometer. Untreated and HIV-1treated cells were used as controls. FIG. 4B displays time-dependentactin depolymerization by Lat-A. Cells were treated with 25 nM Lat-A forvarious times, stained and analyzed as in FIG. 4A. Enhancement of HIV-1latent infection by Lat-A is shown in FIG. 4C. Cells were treated with25 nM Lat-A for 5 minutes, washed, infected with HIV-1 for 5 days, thenactivated with anti-CD3/CD28 bead. As a control, cells were also treatedwith Lat-A for 5 minutes after infection. FIG. 4D shows dose-dependentenhancement of viral replication by Lat-A. Resting CD4 T cells weretreated with different dosages of Lat-A for 5 minutes, then infected andactivated as in FIG. 4C.

At high doses (2.5 μM to 250 nM), Lat-A induced dramatic actindepolymerization, whereas at a lower dose (25 nM) it induced actindepolymerization to an extent similar to that induced by HIV-1 (FIG.4A). Thus, resting T cells were pre-treated with 25 nM Lat-A, theninfected with HIV-1. Enhanced HIV replication by 25 nM Lat-A wasobserved in all donors examined (FIG. 4C).

Importantly, this enhancement was seen only in cells pretreated withLat-A prior to infection (FIG. 4C). These results demonstrate thatartificial depolymerization before infection enhances HIV infection. Theeffects of Lat-A at higher dosages were also tested where Lat-A inducedactin depolymerization was much greater than the physiologicaldepolymerization induced by HIV-1 (FIG. 4D). Interestingly, at 250 nM,donor-dependent variations from enhancement to inhibition occurred (datanot shown). At 2.5 μM, however, Lat-A inhibited viral replication in alldonors (FIG. 4D). These results show that excessive, non-physiologicaldepolymerization can affect T cell function. Thus, besides being abarrier, the cytoskeleton may actively participate in post entryprocesses. Thus excessive depolymerization can affect viral function.

Thus, the stabilization of actin by Jas inhibits HIV replication,whereas depolymerization of actin by Lat-A or CD4/CXCR4 beads enhancesviral replication. Collectively, these findings show that in resting CD4T cells, actin cytoskeleton represents a barrier that needs to bedynamically reorganized to some extent and that HIV-1 exploits the CXCR4signaling pathway to fulfill this requirement.

Example 21 Cofilin Activity in Resting CD4 T Cells and HIV-1 InfectedCells

Given that HIV-1 envelope-CXCR4 signaling mediates actindepolymerization, cofilin activity was compared in resting CD4 T cellsand cells infected with HIV-1.

FIG. 5. shows that HIV 120-CXCR4 signaling triggers P.T-sensitiveactivation of cofilin. Western blots of HIV-1 treated cells were probedfor P-cofilin, then stripped and reprobed for total cofilin (FIG. 5A).FIG. 5B shows activation of cofilin by gp120. Activation of cofilin byanti-CD4/CXCR4 bead (two beads/cell) is shown in FIG. 5C. FIG. 5D showsactivation of cofilin by gp120 promotes its association with actincytoskeleton. F-actin fractions were prepared from gp120 treated restingT cells and immunoblotted for actin (upper band) and cofilin (lowerband). FIG. 5E shows P.T-sensitive activation of cofilin by HIV-1. Cellswere not treated (left panel) or treated (right panel) with P.T theninfected and analyzed as in FIG. 5A. FIG. 5F shows constitutiveactivation of cofilin in transformed cell lines. P-cofilin (upper band)and active cofilin (lower band) was separated by NEPHGE-western blottingand the relative ratio of P-cofilin to active cofilin was indicated atthe bottom. Resting CD4 T cells were used as a control at the right end.F-actin staining of CEM-SS cells infected with HIV-1 is shown in FIG.5G. Cofilin activation induced by PHA plus IL-2 and HIV-1 treatment ofresting CD4 T cells is displayed in FIG. 5H

In resting CD4 T cells, cofilin was largely inactivated byphosphorylation, and upon HIV infection, was activated bydephosphorylation within minutes (FIG. 5A). HIV-induced cofilinactivation was seen in all donors examined. The kinetics correlate wellwith HIV-1 induced actin depolymerization, with strong cofilinactivation and actin depolymerization occurring between 1 to 2 hours(FIG. 2C).

To confirm that cofilin activation was triggered by HIV envelopebinding, resting CD4 T cells were treated with gp120. Again, cofilin wasactivated by gp120 (FIG. 5B). Furthermore, anti-CD4/CXCR4 magnetic beadstimulation of resting CD4 T cells results in cofilin activation (FIG.5D). These data demonstrated that HIV-1 envelope engagement of theCD4/CXCR4 receptors is sufficient to activate cofilin in resting Tcells. To demonstrate the association of active cofilin with F-actin ingp120 stimulated resting T cells, F-actin was fractionated before andafter gp120 treatment. Low level cosedimentation of cofilin with F-actinwas observed in resting T cells (FIG. 5D). Upon gp120 stimulation, therewas a significant increase in cofilin association with F-actin, followedby a decrease in F-actin at 1 hour (FIG. 5D). These data confirm thatactivation of cofilin by gp120 promotes its association with F-actionand subsequent actin depolymerization.

To determine whether cofilin activation is from the Gai dependent CXCR4signaling, resting CD4 T cells were pretreated with P.T and theninfected with HIV-1. Complete inhibition of cofilin activation wasobserved by peltussis toxin (FIG. 5E). This is consistent with P.Tinhibition of actin depolymerization by HIV-1. Thus, HIV envelopebinding to CXCR4 triggers P.T sensitive activation of cofilin and itsassociation with F-actin to promote actin dynamics.

Example 22 Cofilin Activation Mediated by CXCR4Signaling

Contrary to resting CD4 T cells, transformed T cells do not requireCXCR4 signaling to support viral replication. However, binding of gp120to CXCR4 does trigger signaling cascades resulting in Ca²⁺ mobilization(B. J. Doranz et al., J Virol 73, 2752 (April, 1999) and activation ofsignaling molecules such as Pyk2 in cell lines. C. B. Davis et al.,Journal of Experimental Medicine 186, 1793 (1997). Thus, it wasinvestigated if CXCR4 signaling also leads to the activation of cofilinin these cells.

Using nonequilibrium pH gel electrophoresis (NEPHGE) (P. Z. O'Farrell,et al., Cell 12, 1133 (1977)), basal levels of active cofilin weremeasured in transformed cells. In great contrast to resting CD4 T cellsin which cofilin is largely in the inactive form, transformed T cellspredominately carry active cofilin (FIG. 5F). These results show that intransformed cells, cofilin is constitutively active (Y. Samstag et al.,Proc Natl Acad Sci USA 91, 4494 (1994)) and that activation throughCXCR4 is no longer needed for the virus. Consistently, treatment oftransformed CEMSS T cells with HIV-1 did not induce further actindepolymerization (FIG. 5G).

These findings explain previous observations of an unnecessary role ofCXCR4 signaling in HIV-1 infection of transformed cell lines. Forexample, treatment of CXCR4 transfected U87-MG cells with P.T did notaffect viral replication (B. J. Doranz et al., J Virol 73, 2752 (April,1999)). Similarly, several CXCR4 mutants (2333b, 2442, 4442 andCXCR4-QAA) that were not capable of binding SDF-1 or mediating signalingstill supported HIV replication in U87-MG cells (Doranz et al., (1999)).Thus, the requirement for cofilin activation is a unique feature onlyseen in resting T cells and not in transformed cell lines. It also wasobserved that stimulation of resting CD4 T cells with PHA plus IL-2activated cofilin similarly to that of HIV-1 (FIG. 5H). These dataconfirmed that T cell activation can lead to cofilin activationrendering CXCR4 signaling unnecessary for HIV replication.

Example 23 Cofilin Activation is Directly Involved in Viral LatentInfection

In resting CD4 T cells, cofilin is inhibited by phosphorylation atserine-3. The inhibition is maintained through the basal activity of theLIM family protein kinases for which ADF/cofilin proteins are the onlyknown substrates (S. Arber et al., Nature 393, 805 (1998); N. Yang etal., Nature 393, 809 (1998)). To date, there is no known specificinhibitor for LIM kinases.

To demonstrate that activation of cofilin is directly involved in virallatent infection, a synthetic peptide was used to compete with cofilinfor LIMK1 to inhibit cofilin phosphorylation. This peptide, S3, carriesthe N-terminal 16 residues including serine 3 of human cofilin. M.Nishita et al. Molecular & Cellular Biology 22, 774 (2002). Todemonstrate competitive inhibition of cofilin phosphorylation by S3, anin vitro LIMK1 kinase assay was performed using a GST-tagged recombinanthuman cofilin-1 as the substrate.

FIG. 6 illustrates that induction of active cofilin promotes virallatent infection of resting T cells. FIG. 6A illustrates cofilinspecific S3 peptide activates cofilin through competitive inhibition ofcofilin phosphorylation by LIMK1. A recombinant human cofilin was usedas the substrate in the in vitro LIMK1 kinase assay. Q104 was used as acontrol peptide. FIG. 6B illustrates dosage dependent enhancement ofviral replication by S3. Cells were treated with S3 or Q104 for 2 hours,then infected, washed and cultured for 5 days and activated byanti-CD3/CD28 bead. Shown is p24 release at day 8. FIG. 6C shows viralreplication course in cells similarly treated and infected as in FIG.6B. FIG. 6D illustrates that staurosporine induces cofilin activation inresting T cells. Cells were treated with Staurosporine and immunoblottedfor P-cofilin and total cofilin. FIG. 6E illustrates that staurosporineinduces actin depolymerization in resting T cells. Staurosporine inducescofilin activation by direct inhibition of LIMK1, as shown in FIG. 6F.The in vitro LIMK1 kinase assay was performed as described above forFIG. 6A, in the presence or absence of Staurosporine. FIG. 6G showsenhancement of viral replication by Staurosporine. Resting cells weretreated with Staurosporine for 2 hours, infected, washed and incubatedfor 5 days, then activated with anti-CD3/CD28 bead. FIG. 6H shows amodel of gp120-CXCR4 signaling in mediating cofilin activation.

Because it was observed that S3 inhibited cofilin phosphorylation byLIMK1 (FIG. 6A), tests were performed to determine whether activation ofcofilin by S3 would enhance viral replication. Resting CD4 T cells werepre-treated with S3 or a control peptide, Q104, then infected with HIV.S3 enhanced viral replication (FIGS. 6, B and C) and this enhancementwas consistently observed in multiple donors (data not shown). Thus,increasing cofilin activity directly enhances HIV-1 latent infection ofresting CD4 T cells.

Example 24 Kinase Inhibitors Affecting Cofilin Phosphorylation

Given the importance of cofilin for viral latent infection asdemonstrated above, kinase inhibitors were screened for their ability toaffect cofilin phosphorylation in resting T cells. Unexpectedly, it wasdiscovered that staurosporine, a general serine/threonine kinaseinhibitor (T. Tamaoki et al., Biochem Biophys Res Commun 135, 397(1986), had a dramatic inhibition on cofilin phosphorylation. Brieftreatment of resting CD4 T cells with 200 nM staurosporine led togradual dephosphorylation and activation of cofilin in the absence ofany stimulation (FIG. 6D). With the activation of cofilin, actindepolymerization also was observed in staurosporine treated cells (FIG.6E). These results agree with data demonstrating that activation ofcofilin leads to actin depolymerization in resting CD4 T cells (FIG.5D).

To determine whether staurosporine acts on cofilin phosphorylationdirectly, the effect of staurosporine on the kinase activity of LIMK1 invitro using purified human cofilin-1 as the substrate was assayed asdescribed above. At 200 nM, staurosporine directly inhibited thephosphorylation of cofilin by LIMK1 (FIG. 6F). This inhibition was muchstronger than the competitive inhibition by the S3 peptide (FIG. 6A).Thus, staurosporine induces cofilin activation through direct inhibitionon LIMK1. Based on these results, it was further tested whetherstaurosporine would facilitate viral replication. Remarkably, treatmentof resting CD4 T cells with 200 nM staurosporine briefly beforeinfection led to a dramatic enhancement in HIV replication (FIG. 6G).This enhancement was not due to enhancement on T cell activity bystaurosporine.

When resting T cells were similarly treated with staurosporine,incubated and activated, there was a slight inhibition of IL-2 secretionby staurosporine (FIG. 9-10), consistent with previous demonstration ofbroad inhibitory effects of staurosporine on T cell activity. As shownin FIG. 10, resting CD4 T cells were infected with HIV-1, washed, thencultured for 5 days. At day 5, cells were activated by anti-CD3/CD28bead (4 beads per cell) in the presence of 200 nM staurosporine. Viralp24 release was measured. Control cells were identically infected withHIV and activated in the absence of staurosporin.

Additionally, when staurosporine was added during T cell activation atday 5, it inhibited T cell activation and completely abolished HIV-1replication.

Thus, given that staurosporine can cripple a large part of the signalingpathways critical for T cell activity while simultaneously activatingcofilin and greatly enhancing viral replication, activation of cofilinis one of the most critical steps in HIV latent infection of resting Tcells.

Example 25 Cofilin Activation in HIV Positive Patients

The phosphorylation state of cofilin in resting T-cells isolated fromHIV patients was compared with that of healthy donors. As shown in FIG.11, the ratio of active cofilin versus inactive cofilin shifts towardsthe dephosphorylated active form in resting T cells from HIV patients.This is the first report of cofilin dysregulation in a T-cell mediatedimmunodeficiency.

Resting CD4 T cells from five HIV patients on HAART therapy and fivehealthy donors were purified by negative depletion and then lysed andsubsequently immunoblotted for P-cofilin (Ser3) or cofilin. (FIG. 11A).As illustrated in FIG. 11, activation of cofilin occurs in HIV positivepatients. The relative ratio of P-cofilin to cofilin is plotted in FIG.11B. HIV positive donors have statistically significant lower levels ofP-cofilin/cofilin (p=0.001) suggesting higher levels of active cofilin.The results were confirmed by NEPHEGE western blotting using ananti-cofilin antibody. Shown are the absolute ratios of P-cofilin toactive cofilin in three health donors (FIG. 11 C) and three HIV infecteddonors (FIG. 11D).

To control for slight differences in cell number the ratio of P-cofilinto cofilin in each donor was calculated by taking density measurementsof the bands from the western blot using NIH imager software. In HIVpatients, the ratio of P-cofilin to cofilin was significantly lower,with an average of 0.535 compared to 1.142 in healthy donors (p-value0.001). These results suggest that in HIV patients there is a shifttowards activated cofilin compared to healthy donors.

Large scale activation of cofilin in HIV patients is remarkable giventhe fact that a previous report found that there is only 0.2-16.4 HIVlatently infected T cells per 10⁶ resting T cells in patients on HAARTtherapy. Despite this very minimal viral load, global cofilin activationin all the resting T cells purified from HIV patients on HAART therapywas observed. These finding suggest that cofilin activation could notoccur simply as a result of direct HIV infection but is the result ofindirect mechanisms perhaps through either viral or cellular gp120shedding. In fact, it's been previously shown that gp120 treatment ofresting CD4 T cells in vitro at concentrations below 50 nM result incofilin dephosphorylation and subsequent activation.

The implications of cofilin activation in resting T cells in HIVpatients are numerous. Currently, the only diagnostic markers for HIVdisease progression are viral load numbers as calculated by PCR andtotal CD4 T cell count. It is widely accepted that there is a bystandereffect of HIV-1 infected T cells on other non-infected cells, givingrise to apoptosis, anergy, and impaired T cell homing. However, there isno current way to quantify this T-cell change. The fact that cofilinplays a critical role in T cell activation, chemotaxis and, appears tobe dysregulated in HIV patients, means cofilin activation could serve asa clinical marker of HIV disease progression.

Example 26 Inhibition of HIV-1 infection of resting CD4 T cells

We treated resting CD4 T cells with a variety of inhibitors for 2 hoursbefore infection, then infected them with HIV-1 for 2 hours. Afterinfection, cells were washed twice to remove the inhibitors and thecell-free virus. We incubated infected cells for 5 days, then activatedthe cells by adding anti-CD3/CD28 antibody coated magnetic beads (4beads per cells). Then, we monitored viral replication daily from day 5to 9. The results are depicted in FIGS. 13-15, which shows viralreplication at day 8. The Y-axes show viral replication judged byrelease of viral p24 protein into the medium (pg/ml in p24concentration).

TABLE 2 SEQ ID NO: Source Sequence 1 Homo   atggcctccggtgtggctgtctctgatggtgtca sapienstcaaggtgttcaacgacatgaaggtgcgtaagtc mRNA forttcaacgccagaggaggtgaagaagcgcaagaag cofilin,gcggtgctcttctgcctgagtgaggacaagaaga completeacatcatcctggaggagggcaaggagatcctggt cds; gggcgatgtgggccagactgtcgacgatccctac GenBankgccacctttgtcaagatgctgccagataaggact gi: 219544gccgctatgccctctatgatgcaacctatgagac caaggagagcaagaaggaggatctggtgtttatcttctgggcccccgagtctgcgccccttaagagca aaatgatttatgccagctccaaggacgccatcaagaagaagctgacagggatcaagcatgaattgcaa gcaaactgctacgaggaggtcaaggaccgctgcatccctggcagagaagctggggggcagtgcggtca tctccctggagggcaagccttgtga 2 Homo  masgvavsdgvikvfndmkvrksstpeevkkrkk sapiensavlfclsedkkniileegkeilvgdvgqtvddpy cofilin atfvkmlpdkdcryalydatyetkeskkedlvfi proteinfwapesaplkskmiyasskdaikkkltgikhelq sequenceancyeevkdrctlaeklggsavislegkpl GenBank gi: 219545

1. A method for treating and/or preventing HIV infection in a patient,comprising: administering to said patient an agent that inhibits HIVtrigger receptor signaling, inhibits actin depolymerization; enhancesthe assembly of actin; stabilizes actin filaments; inducespolymerization of monomeric actin; binds to F-actin or cofilin; orinhibits actin and cofilin activity.
 2. The method of claim 1, whereinsaid agent is selected from the group consisting of jasplakinolide,PD59, FK506; wortmannin, LY294002, PP2, PP2A, PP1, AG1478, AG1296;slingshot phosphatases, FR225659, fostriecin, calyculin A, cantharidin,jasplakinolide; phaloidin; chondramides, chondramide A, B, C, and D;(−)-doliculide; dolastatin-11; dolastatin 3-Nor; majusculamide;dolastatin Hmp; alpha-cyano-3,4-dihydroxy-N-benzylcinnamide (AG490);1,2,3,4,5,6-; JSI-124; benzylidenemalonitriles (“tyrphostins”);WHI-P154; WHI-P151; pyrrolo[2,3-d]-pyrimidines;benzimisazo[4,5-f]isoquinolinone derivatives; AG1801; WP1034; WP1050;WP1015; WP1-1066; WP1129; WP1130; WP1119; WP1026; WP1127; JSI-124;cucurbitacin I; cucurbitacin A; cucurbitacin B; cucurbitacin D;cucurbitacin E; tetrahydro-cucurbitacin I; PD98059(2′-amino-3′-methoxyflavone); UO126; SL327; olomoucine;5-iodotubercidin; arctigenin; 4-bromo or 4-iodo phenylaminobenzhydroxamic acid derivatives; N3 alkylated benzimidazole derivatives;FR225659; fostriecin; Calyculin A; okadaic acid; cantharidin;TCM-platinum agents containing demethylcantharidin; genistein; MEKinhibitor, and derivatives thereof.
 3. The method of claim 1, whereinsaid agent inhibits the JAC2 signaling pathway.
 4. The method of claim1, wherein said agent inhibits the tyrosine kinase signaling pathway. 5.The method of claim 1, wherein said agent is a phosphatase inhibitor. 6.The method of claim 1, wherein said agent inhibits the Rac/Pac1/Lmksignaling pathway.
 7. The method of claim 1, wherein said agent inhibitsthe signal transduction activity of CXCR4.
 8. A method for identifyingcompounds that inhibit HIV infection, comprising evaluating a compound'sability to alter the phosphorylation state of a protein of theADF/cofilin family.
 9. The method of claim 8, wherein the evaluationcomprises determining whether the compound can inhibit thedephosphorylation of serine-3 residue of cofilin.
 10. A compositioncomprising: a) an effective amount of a compound selected from the groupconsisting of: jasplakinolide, PD59, FK506; wortmannin, LY294002, PP2,PP2A, PP1, AG1478, AG1296; slingshot phosphatases, FR225659, fostriecin,calyculin A, cantharidin, jasplakinolide; phaloidin; chondramides,chondramide A, B, C, and D; (−)-doliculide; dolastatin-11; dolastatin3-Nor; majusculamide; dolastatin Hmp;alpha-cyano-3,4-dihydroxy-N-benzylcinnamide (AG490); 1,2,3,4,5,6-;JSI-124; benzylidenemalonitriles (“tyrphostins”); WHI-P154; WHI-P151;pyrrolo[2,3-d]-pyrimidines; benzimisazo[4,5-f]isoquinolinonederivatives; AG1801; WP1034; WP1050; WP1015; WP1-1066; WP1129; WP1130;WP1119; WP1026; WP1127; JSI-124; cucurbitacin I; cucurbitacin A;cucurbitacin B; cucurbitacin D; cucurbitacin E; tetrahydro-cucurbitacinI; PD98059 (2′-amino-3′-methoxyflavone); UO126; SL327; olomoucine;5-iodotubercidin; arctigenin; 4-bromo or 4-iodo phenylaminobenzhydroxamic acid derivatives; N3 alkylated benzimidazole derivatives;FR225659; fostriecin; Calyculin A; okadaic acid; cantharidin;TCM-platinum agents containing demethylcantharidin; genistein; MEKinhibitor, and derivatives thereof; b) an effective amount of ananti-retroviral agent; and c) a pharmaceutically acceptable excipient.